Abstract: Ammonium thiosulfate as a novel sulfiding agent was used for presulfidation of hydrodesulfurization (HDS) catalysts. The presulfided Mo/Al₂O₃ catalysts with different molar ratios of S/Mo were prepared by impregnating Mo/Al₂O₃ catalysts with an aqueous solution of ammonium thiosulfate, followed by drying. Their characteristics of crystal phases, activation, and surface compositions were examined by X-ray diffraction, thermogravimetric analysis, and X-ray photoelectron spectroscopy. The S^2- and S⁶⁺ in ammonium thiosulfate play different roles during the activation of presulfided catalysts. The S^2- sulfides Mo and S⁶⁺ reacts with Al₂O₃ support, decreasing the interaction of support with the active metal. The collaboration between S^2- and S⁶⁺ ions improves the sulfidation of MoO₃. The best HDS performance was observed for the presulfided catalyst with S/Mo = 3. Its thiophene HDS activity is 17% higher than that of Mo/Al₂O₃ catalyst sulfided with H₂S.